It has become increasingly clear that, in vivo, RNA processing events occur co-transcriptionally, mediated by RNA processing factors associated with the carboxy-terminal domain (CTD) of the elongating RNA pol II complex. It would be expected that viral regulators of gene expression would influence this process, however, few examples of such viral regulators have so far been identified. We have recently demonstrated that in the presence of adenovirus type 5 (Ad5), the large Rep protein of adeno-associated virus type 2 (AAV2), a member of the Parvoviridae Family, acts as a co-transcriptional trans-regulator of RNA processing. We propose to further characterize this novel activity of Rep in Specific Aim I, by: 1.determining the cis-acting signals in the AAV2 transcription unit that are required and sufficient to be responsive to the RNA processing effects of Rep and Ad5; 2.determining the trans-acting helper functions that are required to support Rep's effect, and by determining the domains of the Rep protein that mediate its RNA processing effect; and 3.determining the mechanism of Rep's co-transcriptional effect on processing of AAV2 RNA. Specifically, we will test our model that Rep alters the composition of RNA processing factors associated with the CTD of the elongating transcription complex. Our survey of the transcription profile of the other AAV serotypes has revealed that RNA produced from AAV5 efficiently utilizes a polyadenylation site, (pA) p, within the viral intron. The usage of (pA) p is directly proportional to the distance between the promoter and that site, and (pA) p is subject to inhibition by an upstream intron donor. This will be further characterized in Specific Aim 2, by: 1.characterizing the cis-and trans-acting determinants that govern the alternative polyadenylation of AAV5 RNA; 2.determining why RNAs generated from the upstream P7 promoter utilize (pA) p, and those from nearby P41 read through; and 3.examining the consequences of internal polyadenylation for the AAV5 life cycle. These aspects of gene expression are critically important features of AAV biology. Also, as both of these RNA processing functions are probably best understood in a co-transcriptional framework, their characterization in the tractable AAV system will help illuminate this new paradigm of gene expression. [unreadable] [unreadable]